Humans often consume alcoholic beverages to promote social interactions. On the other hand, alcohol abuse can have devastating effects on long-term social attachments. The mechanisms alcohol's effects on social relationships have been difficult to address. The situation changed with studies showing that many features of social bonding can be modeled using socially monogamous prairie voles (Microtus ochrogaster). Mechanisms regulating pair bonding in voles have later been shown to contribute to human social bonding, indicating strong construct validity of this animal model. We have initiated studies analyzing effects of social environment on alcohol consumption and, more recently, identified striking sex-dependent effects of voluntary alcohol consumption on the formation of male-female bonds in prairie voles. Specifically, voluntary alcohol consumption inhibited formation of partner preference in male animals, but facilitated partner preference in females. These findings indicated that alcohol acts through biological mechanisms to produce specific effects on adult social attachments. In parallel we found that alcohol drinking during cohabitation also produces sex-specific effects on c-Fos expression in the centrally-projecting Edinger-Westphal nucleus (EWcp). This application proposes to test whether EWcp contributes to alcohol's effects on pair bonding and further investigate alcohol's effects on pair bonding in three Specific Aims (SAs). In SA1 we will use CLARITY-based and standard immunohistochemistry to measure levels of several peptides and c-Fos across different brain regions during cohabitation in the presence or absence of alcohol. In SA2 we will test the causal role of EWcp activation in alcohol's effects on partner preference using the Designer Receptor(s) Exclusively Activated by Designer Drug(s) approach. Subsequent studies will test the roles of specific subpopulations of EWcp or brain regions identified in SA1. Finally, while our previous studies found effects of the first day of voluntary alcohol drinking on formation of pair-bonds, in SA3 we will test alcohol's effects on pair bonding following prolonged alcohol use and alcohol's effects on already established pair bonds.